The present invention relates to salmonella vaccines that are useful against Salmonellosis caused by heterologous serotypes of Salmonella in mammals. More specifically, the invention relates to salmonella vaccines for swine incorporating Salmonella cholerasuis which provides cross protection against disease caused by heterologous Salmonella species including but not limited to Salmonella typhimurium. 
Salmonellosis of swine is one of the most economically important of the enteric and septicemic diseases affecting young pigs. Although many serotypes of Salmonella have been isolated from pigs, S. cholerasuis var kunzendorf and S. typhimurium are the two most frequently isolated serotypes associated with clinical disease. S. cholerasuis is host-adapted to swine and most often causes fatal septicemic disease with little involvement of the intestinal tract. On the other hand, S. typhimurium typically causes enteroinvasive disease characterized primarily or exclusively by diarrhea. The initial signs of the disease include watery, yellowish diarrhea without mucin or melena. Affected pigs often exhibit anorexia, lethargy, and fever ranging from 105 to 107 degrees Fahrenheit. Mortality is usually low and occurs only after several days of diarrhea, presumably the result of hypokalemia and dehydration. Literature clearly notes that both the type of infection and host range vary significantly between S. cholerasuis and S. typhimurium. 
It has been known to use Salmonella cholerasuis vaccines such ARGUS SC(trademark) vaccine (Intervet Inc., Millsboro, Del.) to protect pigs against diseases caused by infection from S. cholerasuis (homologous protection). The vaccine of the present invention incorporates a modified live S. cholerasuis, the composition of which is described generally in U.S. Pat. No. 5,468,485. More specifically, the patent discloses a vaccine for the immunization of vertebrates or invertebrates comprising an avirulent derivative of S. cholerasuis. The derivative is substantially incapable of producing functional adenylate cyclase (cya gene deletion) and/or cyclic AMP receptor protein (crp). The patent also discloses a vaccine for immunization of a vertebrate or invertebrate comprising a virulent derivative of a pathogenic microbe, which is substantially incapable of producing functional adenylate cyclase and/or cyclic AMP receptor protein. Said pathogenic microbe is capable of expressing a recombinant gene derived from a pathogen of said vertebrate to produce an antigen capable of inducing an immune response in said vertebrate against said pathogen. This patent describes construction of various avirulent Salmonella species but does not disclose or claim use of a S. cholerasuis vaccine to protect pigs against disease caused by a heterologous Salmonella such as S. typhimuirum. 
U.S. Pat. No. 5,804,194 discloses vaccines containing Salmonella bacteria attenuated by mutation of the HTRA gene. This mutation also produces avirulent salmonella vaccines which appear to be safe when injected into mice. Also described is vaccination of mice by a vaccine of the invention followed by challenge with a homologous S. typhimurium strain. There is no description or claim of a S. cholerasuis vaccine having the capability to cross protect against diseases caused by heterologous Salmonella species.
U.S. Pat. No. 5,843,426 discloses salmonella vaccines containing salmonella organisms, the virulence of which is attenuated by a deletion of a portion of the PhQ gene and Salmonella organisms having a deletion of both the PhQ gene and the PhoP gene. There is no mention of a S. cholerasuis vaccine which can cross protect pigs against disease caused by heterologous Salmonella species.
Miller et al., 1989, Proc. Natl. Acad. Sci USA 86:5054 discloses S. typhimurium strains with mutations in the positive regulatory regulon phoP which are markedly attenuated in virulence for BALB C mice. The phoP regulon is composed of two genes present in an operon termed phoP and phoQ. The PhoP and the phoQ gene products are highly similar to other members of bacterial two-component transcriptional regulators that respond to environmental stimuli and control the expression of a large number of other genes. A mutation at one of these PhoP regulatory regions, regulates the pagC genes and produces a virulence defect. Strains with pagC, phoP and phoQ mutations afford partial protection to subsequent challenge by wild-type S. typhimurium. However, there is no description or claim made for S. cholerasuis vaccines which cross protect against diseases caused by heterologous Salmonella species.
U.S. Pat. No. 5,436,001 discloses methods of attenuating virulent Gram negative bacteria in order to produce avirulent vaccine strains. The method is described as serial passaging a gram negative organism through phagocytic cells a sufficient number of times until the bacteria are rendered avirulent to the animal host while still being immunogenic. A method to attenuate S. cholerasuis var, Kunzendorf strain 38 is described. Several pig vaccination/challenge studies were conducted. These studies demonstrated that a S. cholerasuis attenuated, and produced according to the methods of the patent, could protect against a homologous S. cholerasuis challenge. However, there was no description or claim of cross protection using a S. cholerasuis vaccine to protect against disease caused by a heterologous Salmonella such as S. typhimurium. 
Smith et al (Am J Vet Res, 1984, Vol 45, No. 11: 2231-2235) describes an aromatic-dependent avirulent S. dublin strain which was tested for safety as a parenteral vaccine for calves as well as for its capability to protect calves from challenge with homologous S. dublin or heterologous S. typhimurium. Indeed, the vaccine was shown to be safe and provided protection against disease in cattle caused by both S. dublin and S. typhimurium. However, the publication states that in a previous study, conducted in an identical manner, protection was not produced. Additionally, it is noted that this publication does not describe the use of S. cholerasuis vaccines to cross protect against disease caused by eterologous Salmonella of swine nor does it describe oral vaccination.
Alternately, Smith et al (Am J Vet Res, 1984, Vol 45, No 11: 858-1861) describe the production of an aromatic-dependent avirulent S. typhimurium which was tested for safety and efficacy in calves. Both the ability to protect against a homologous S. typhimurium challenge and a heterologous S. dublin challenge were evaluated. The parental vaccine was found to be relatively safe although some disease signs were noted post vaccination. Since 2 of 5 vaccinated calves had slight anorexia, 4 of 5 had diarrhea, and all had marked febrile response after challenge exposure, it was determined that the aromatic-dependent avirulent S. typhimurium vaccine did not protect calves against a different serotype (S. dublin) as well as it had against a homologous serotype (S. typhimurium). However, even against the homologous challenge, 3 of 7 vaccinated calves developed mild diarrhea and 1 of 7 calves had a positive blood culture. This publication actually teaches away from the present invention of a S. cholerasuis vaccine which cross protects against disease caused by a heterologous Salmonella species such as S. typhimurium. 
Fox et al (Am J Vet Res, 1997, Vol 58, No. 3, 265-271) describes an attempt to use an avirulent live S. cholerasuis vaccine to protect calves against disease caused by S. dublin infection. The vaccine, designated SC54, contains an avirulent live culture of S. cholerasuis normally used for intranasal or oral vaccination of swine to aid in the prevention of salmonellosis in swine caused by S. cholerasuis. The results indicate a varied response to vaccination of calves with different doses and routes of administration of SC54. Vaccination with SC54 did not prevent the fever and fecal shedding of S. dublin but did reduce the bacteremia and frequency of S. dublin recovery from organs of calves at necropsy when vaccine was administered intranasally. The conclusion was that SC54 appears to have potential as a safe and effective vaccine against disease in calves caused by S. dublin. However, its efficacy against S. typhimurium infection in calves was not demonstrated. This publication teaches away from use of a S. cholerasuis vaccine for cross protection of swine against disease caused by a heterologous Salmonella species such as S. typhimurium. 
Letellier et al (1999 ISECSP: Production Intervention) has described the evaluation of different treatments to reduce Salmonella infections in swine. These treatments include Salmonella cholerasuis vaccines FOS (1% in feed, Encore Technologies), FERLAC-2(trademark) in feed, (Rosell Institute, Montreal Canada) and intranasal vaccination with SC54(trademark) (Boehringer Ingleheim, Iowa). The publication indicates that the colonization of mesenteric lymph nodes for SC54(trademark) vaccinated pigs was reduced in comparison to the control group. There was also a reduction in prevalence of Salmonella in the ileum. However, there was no difference observed in quantitative evaluation of S. typhimurium in the mesenteric lymph nodes. Also, phagocytosis from whole blood phagocytes was not increased after treatments suggesting that systemic stimulation of phagocytes was not sufficient to increase resistance to S. typhimurium. This publication, although testing an avirulent S. cholerasuis vaccine for cross protection in pigs was not successful in demonstrating such cross protection. It teaches away from the present invention.
Letellier et al (1999 ISECSP: Production Intervention) reported on the Assessment of different treatments to reduce Salmonella in swine. In this study, SC54(trademark) was again evaluated for potential cross protection of swine against disease caused by hetero-logous Salmonella. Following experimental infection with S. typhimurium, 70% of the control pigs became colonized by S. typhimurium in the gut and 60% were infected in mesenteric lymph nodes. No significant difference was observed in quantitative evaluation of S. typhimurium in the mesenteric lymph nodes in the different groups (including the SC54(trademark) group). SC54(trademark) did reduce the presence of S. typhimurium in the mesenteric lymph nodes but it did not reduce shedding of S. typhimurium in feces. In fact, there was an increase in the fecal shedding in the vaccinate group as compared with the control group. This publication does not present conclusive evidence and does not teach cross protection of heterologous Salmonella by vaccination with a S. cholerasuis vaccine.
While it has been generally known that there can be cross reactivity (immunological tests indicate that antibodies of one serotype of Salmonella react with other serotypes of Salmonella), cross protection between Salmonella serotypes (heterologous protection) has been poorly characterized and unproven. These showings are insufficient for making and using salmonella vaccines incorporating one serotype to provide protection in swine against another. Certainly, the use S. cholerasuis vaccine which is typically useful in protecting pigs against septicemia and death would not be expected to protect against S. typhimurium which produces a severe diarrhea in all mammals.
By this invention, Applicant has provided an avirulent S. cholerasuis vaccine, which can cross protect against disease caused by infection with heterologous Salmonella serotypes such as S. typhimurium. 
As set forth above, the present invention relates to a method of protecting pigs against disease caused by infection with heterologous serotypes of Salmonella (cross protection) including but not limited to S. typhimurium comprising administering to the pigs a modified live vaccine incorporating S. cholerasuis. It is a distinct feature of the invention that the vaccine can be administered orally or parenterally. It is also a distinct feature that the vaccine can be mass delivered in drinking water.
The avirulent S. cholerasuis vaccine of the present invention comprises a S. cholerasuis strain "khgr"3781 (xcex94 cya xcex94(crp-cdt) which is a mutant with portions of the genes encoding adenylate cyclase (cya), cAMP receptor protein (crp) and the ability to colonize deep tissue (cdt) modified by transposon mutagenesis. In addition, the organism contains an auxotrophic requirement for methionine. Therefore, the production of the vaccine requires that the S. cholerasuis avirulent strain be grown on media supplemented with methionine. After growing to an optimum optical density or to a count (colony forming units)  greater than 2xc3x97108 the culture is harvested, stabilizer is added and the stabilized culture is filled into final containers and lyophilized (freeze-dried). Stabilizers can be of any type including but not limited to sucrose, gelatin, skim milk, dextrose, Lactalbumin Hydrolysate, NZ amine, Glutamic Acid and combinations thereof. A preferred stabilizer is a combination of 3.0% NZ-amine Type AS, 0.3% Glutamic Acid, monopotassium salt, 25% sucrose, 0.2% Lactalbumin Hydrolysate (Edamin S) and 5.0% Gelatin. At the time of use, diluent is added to the lyophilized final product container to resuspend the S. cholerasuis modified live vaccine and the vaccine is administered to pigs. Administration can be 1.0 mL delivered parenterally to pigs (subcutaneously, intramuscularly or intraperitoneally), 1.0 mL delivered to a mucous membrane such as into the mouth, eye, vagina, or rectum, or preferably, addition of 1.0 mL per pig to a water proportioner such that pigs will drink the water and be vaccinated simultaneously. The specific vaccine of the present invention, Argus SC(trademark), is used as an aid in prevention of pneumonia, diarrhea, septicemia and mortality caused by S. cholerasuis in pigs 3 weeks of age or older.
As would be realized from the foregoing, it will be within the purview of the skilled artisan to make and use a modified live S. cholerasuis vaccine such as Argus SC(trademark). The S. cholerasuis is grown in a media selected for optimization of the production of immunogenic avirulent organisms. Optimal growth of S. cholerasuis strain "khgr"3781 (xcex94 cya xcex94(crp-cdt) is reached when the culture demonstrates an optical density of 2.5 at 600 nm. This equates to approximately 2xc3x9710 8 CFU/mL. However, a dose as low as 1xc3x97105 has been shown to be effective. The preferred dose is at least 1xc3x97107 CFU/mL. The term immunogenic avirulent organisms means that the organisms will not produce disease when administered to pigs (they are safe) and that when the organisms are formulated into a vaccine, the vaccine will cross protect swine from diseases caused by homologous and heterologous serotypes of Salmonella including but not limited to S. typhimurium. After growth of the S. cholerasuis it is generally mixed with a stabilizer selected from the group consisting of NZ-amine, Glutamic Acid, Sucrose, Lactalbumin hydrolysate, gelatin, other stabilizing sugars and combinations thereof and then dried. Drying can be accomplished by lyophilization (freeze-drying), vitrification, glassification, or any other method which retains the viability of the organisms. The vaccine can be administered by any route selected from the group consisting of oral, drinking water, intranasal, intramucosal, intramuscular, subcutaneous, intravenous and intraperitoneal. The preferred route is oral or intranasal via drinking water.